Worldwide elimination of HIV transmission from mother-to-child is theoretically achievable. In Cambodia, antenatal care (ANC) prevalence has dropped from 1.6% (2003) to 0.71% (2009). However, success in minimizing vertical transmission has been limited by low testing uptake at ANC and delivery. We trained midwives in counselling and performance of an HIV rapid test, incorporated point-of-care testing into routine antenatal and maternity services and determined acceptability, feasibility, accuracy, cost and yield after one year. In all, 97.3% of ANC clients and 73.0% of maternity admissions had unknown HIV status. Testing was offered to 97.6% and 95.0% of untested ANC and maternity clients, respectively. Acceptance rates were 95.5% and 99.4%. Partner testing rate was 38.6%. HIV was diagnosed in 0.1% of ANC clients, 0.4% of partners and 0.9% of women at delivery. For an operational district with an average population of 158,000, point-of-care testing was estimated in one year to identify 19 HIV-infected pregnant women, nine men, 14 discordant couples and 16 exposed infants who otherwise would have not received prophylaxis. Cost was less than $3.75 per person tested. Point-of-care testing during ANC and at delivery is feasible, acceptable and contributes to reducing mother-to-child transmission.